plasticity of 150-loop in influenza neuraminidase explored ... · plasticity of 150-loop in...

Plasticity of 150-Loop in Influenza NeuraminidaseExplored by Hamiltonian Replica Exchange MolecularDynamics SimulationsNanyu Han, Yuguang Mu*

School of Biological Sciences, Nanyang Technological University, Singapore, Singapore

Abstract

Neuraminidase (NA) of influenza is a key target for antiviral inhibitors, and the 150-cavity in group-1 NA provides newinsight in treating this disease. However, NA of 2009 pandemic influenza (09N1) was found lacking this cavity in a crystalstructure. To address the issue of flexibility of the 150-loop, Hamiltonian replica exchange molecular dynamics simulationswere performed on different groups of NAs. Free energy landscape calculated based on the volume of 150-cavity indicatesthat 09N1 prefers open forms of 150-loop. The turn A (residues 147–150) of the 150-loop is discovered as the mostdynamical motif which induces the inter-conversion of this loop among different conformations. In the turn A, thebackbone dynamic of residue 149 is highly related with the shape of 150-loop, thus can function as a marker for theconformation of 150-loop. As a contrast, the closed conformation of 150-loop is more energetically favorable in N2, one ofgroup-2 NAs. The D147-H150 salt bridge is found having no correlation with the conformation of 150-loop. Instead theintimate salt bridge interaction between the 150 and 430 loops in N2 variant contributes the stabilizing factor for the closedform of 150-loop. The clustering analysis elaborates the structural plasticity of the loop. This enhanced sampling simulationprovides more information in further structural-based drug discovery on influenza virus.

Citation: Han N, Mu Y (2013) Plasticity of 150-Loop in Influenza Neuraminidase Explored by Hamiltonian Replica Exchange Molecular Dynamics Simulations. PLoSONE 8(4): e60995. doi:10.1371/journal.pone.0060995

Editor: Pratul K. Agarwal, Oak Ridge National Laboratory, United States of America

Received December 17, 2012; Accepted March 5, 2013; Published April 10, 2013

Copyright: � 2013 Han, Mu. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricteduse, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: This work is partially supported by the IDA Cloud Computing Call for Project Proposals 2012 and MOE NTU Tier 1 grant RG23/11. The funders had norole in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing Interests: The authors have declared that no competing interests exist.

* E-mail: [email protected]

Introduction

Influenza virus causes a great threat to people when it emerges

as pandemic through reassortment during coinfection of different

host species [1]. Pandemic influenza has high morbidity and

mortality rates due to lack of prior immunity in humans [2], [3].

After adapted to humans, the seasonal influenza virus with high

mutation rate still impacts public health [4]. In order to prevent

and control the influenza virus infections, two strategies could

apply: vaccines and antiviral drugs. It takes three to six months to

create a vaccine in treating a newly emerged virus strain. During

this period, the novel strain can spread globally, infect human and

cause great damage to the economy [5]. In this lag phase, taking

antiviral drugs is the only available approach in controlling and

stopping influenza infections. In addition, because influenza virus

infection cannot be completely prevented by vaccination, antiviral

drugs are still necessary for the therapeutic treatment of influenza

[6].

Neuraminidase (NA), which functions by cleaving the sialic acid

on the host cells and facilitating viruses shedding, is an ideal drug

target [7]. Currently, four anti-NA drugs have been approved:

Oseltamivir [8], Zanamivir [9], Peramivir [10], and Laninamivir

[11]. In 2006, NAs were found to be divided into two groups based

on phylogenetic distinction, group-1 (N1, N4, N5, N8), group-2

(N2, N3, N6, N7, N9) [12]. Historically, Oseltamivir and

Zanamivir were developed based on group-2 NA structures,

which was a successful demonstration of the rational structure-

based drug development strategy [13]. A recent crystal structure of

a group-1 NA contains a cavity (150-cavity) adjacent to the active

site which can be exploited to develop new anti-influenza drugs

[12], [14]. The 150-cavity is capped by 150-loop which is

composed of six residues from 147 to 152, and the sequence of

150-loop is relatively conserved in different sub-groups of

influenza virus. It is noteworthy that the presence of 150-cavity

is a character for group-1 NA, so that the 150-cavity in group-1

NA provides new opportunity in defeating influenza virus.

Interestingly, a crystal structure of NA in 2009 pandemic H1N1

(09N1) revealed a deficient 150-cavity which is different from

structures of other group-1 NAs [15]. Therefore, several questions

need to be addressed: Could the new drug that specifically

targeting the 150-cavity be effective on all group-1 influenza

viruses? Could the 150-loop conformation of 09N1 inter-convert

between the open and closed conformations? Which conformation

of 150-loop is more energetically favorable in group-1 NAs? One

following research discovered that the 150-loop of 09N1 prefers to

exhibit in an open conformation based on normal MD study [16].

Some other works also revealed that the 150-loop conformation of

group-1 NAs could exert an even wider extended 150-cavity in the

simulation [17], [18], [19]. In order to extensively explore the

heterogeneity of the loop conformation and provide a global free

energy landscape of the 150-loop dynamics, we performed

Hamiltonian replica exchange molecular dynamics (HREMD)

PLOS ONE | www.plosone.org 1 April 2013 | Volume 8 | Issue 4 | e60995

simulations on 09N1 [15]. For comparison, one of group-2 NAs

was also included in the study [20].

In this enhanced sampling method, HREMD, Hamiltonians

except one replica were modified by increasing the van der Waals

repulsion forces acting only within a selected group of atoms of the

protein. The potential energy barriers for breaking favorable

contacts within the selected atoms, such as hydrogen bonds,

hydrophobic interactions, can be gradually reduced in the

modified Hamiltonians. Meanwhile the majority of the interac-

tions in the system, such as, protein-water, water-water interac-

tions and the interaction within other part of the protein

(excluding the selected group), keep intact, rendering the number

of replicas needed significantly lower than that of the standard

temperature REMD (TREMD) [21]. Because in TREMD,

escaping from free energy local minima works through increasing

temperature that influences all degrees of freedom of the system.

Comparing with normal MD at room temperature, HREMD

reduces the energy barrier formed within the selected group of

atoms, helps the system to escape from meta-stable states quickly.

Our previous research demonstrated its efficiency and accuracy in

comparison with TREMD and normal MD [21], [22]. Here,

HREMD simulations were performed on 09N1 NA with two

different initial conformations, one being a closed 150-loop, the

other being a modified open 150-cavity. For comparison, one of

group-2 NAs, N2, was also included in the HREMD simulations.

In all systems, Hamiltonian potentials were modified on 9 residues

in NAs (residues 145–153) including the 150-loop (147–152). The

free energy landscape of the 150-loop was extensively explored,

and different states of 150-loop such as open, closed, intermediate

conformations as well as widely-open configurations were all

sampled in the simulations.

Methods

Simulation methodThe potential of the system is composed of

E0(X )~Epp(X )zEpw(X )zEww(X );

where the Epp, Epw, and Eww are the protein internal energy, the

interaction energy between protein and water, and the interaction

of the water molecule, respectively. In HREMD simulation, we

only modify the Hamiltonian of Epp(X) term. So the Epp(X) can be

further decomposed as

Epp(X )~Ebondedpp zEele

pp zEvdWpp :

Ebondedpp and Eele

pp are bonded and electrostatic interaction within

the protein, respective. The van der Waals terms have two

components

EvdWpp ~

Xivj

C12,ij

r12ij

{X

ivj

C6,ij

r6ij

~EvdWpp,12{EvdW

pp,6 :

One is attractive term, EvdWpp,6 , and the other is repulsive term,

EvdWpp,12. In our HREMD simulation, the repulsive term will be

modified as

Em(X )~E00(X )zlmEvdW

pp,12;

and

E0o(X )~Epw(X )zEww(X )zEbonded

pp zEelepp {EvdW

pp,6 ;

where E00(X ) is the unmodified potential energy excluding the

repulsive van der Waal interactions, and lm value is an adjustable

parameter. When lm is set to 1, Em(X) = E0(X). With lm value is

larger than 1, the repulsive forces between atoms of protein will be

enhanced. The replica exchange acceptance probability follows

Metropolis criteria. Detail information can be found in Ref. 21.

Simulation systemsThree independent systems were set up. In the first system,

conformation of the NA was taken from the crystal structure 3NSS

for A/California/04/2009 (09N1). In this structure the 150-loop is

in the closed conformation, thus, this system is abbreviated as N1c

system [15]. In order to get efficient sampling and to speed up the

convergence of simulations, the same structure of 09N1 NA with

an open 150-cavity was build through SWISS-MODEL sever [23]

based on the structure of 2HTY for A/Vietnam/1203/04 [12],

abbreviated as N1o system. Recently, a mutant 09N1 crystal

structure with an open 150-cavity were published [24]. After pair

wise alignment and superimposing all a carbon atoms of the

modeled N1o structure onto the open-state 09N1 crystal structure,

an all atom RMSD value of 0.292 A was obtained. This small

RMSD value indicates that the modeled N1o structure is very

similar to the crystal structure, and provides validation of the

initial open-state structure used in the HREMD simulation. One

of group-2 NAs, with pdb entry 1NN2 for A/Aichi/3/67 (N2) was

chosen for the third system, to compare change of loop

conformation between different sub groups of NAs [20]. All

systems were set up with the same procedures. The NA in each

system was solvated in an octahedron box with TIP3P water [25],

and the minimal distance between the protein and the edge of the

box was set to 0.8 nm. Sodium and chloride ions were added with

a concentration of 100 mM to neutralize the system. Protonation

states for histidines were determined by Chimera software [26].

The GROMACS [27] program suite version 4.5.5 and Am-

ber99SB force field [28] were used in all the simulations. The

details of the simulation systems can be found in Table 1.

Each HREMD simulation was performed with six replicas at

T = 300 K. The Hamiltonian potential was modified on 9 residues

including the 150-loop in NAs (145–153). The values of lm were 1,

1.05, 1.11, 1.17, 1.23, and 1.3 [21]. The values of lm were chosen

to achieve nearly equal exchange acceptance ratio between

neighboring replicas which was averaged around 30%. All the

simulations ran for 300 ns per replica in an isothermal-isobaric

ensemble (300 K, 1 bar), and 5 ns normal MD equilibration

simulations were performed before HREMD simulations with all

heavy atom of protein restricted. Bond length constrain was

applied to all bonds that contain hydrogen based on LINCS

protocol [29]. Therefore, an integration step of 0.002 ps is allowed

in simulations. Each replica was set to attempt exchanging at every

500 integration steps (1ps). Electrostatic interactions were treated

with Particle Mesh Ewald method with a cutoff of 9 A, grid

spacing for the FFT grid ,1.2 A [30]. The cutoff 12 A was used in

the calculation of van der Waals interaction. GROMACS 4.5.5

Plasticity of 150-Loop in NA Explored by HREMD


source code was modified by our group in the exchanging step

when perform detailed balance. The modified source code will be

available upon request.

Analysis methodRoot mean squared deviation (RMSD)

calculation. RMSD of 150-loop was calculated by superimpos-

ing the protein excluding the 150-loop to reference structure. Here

the reference structure is the crystal NA structure with an open or

closed conformation of 150-loop, and all the calculated snapshots

are from H0 trajectory, in which no modified Hamiltonian

potential is applied.

Volume of 150-cavity calculation. The volume of 150-

cavity was calculated using a python script called POVME [31].

First, a 3D-grid was built fully covering the 150-cavity of a

reference open structure of NA, i.e., a crystal PDB structure with

open form of 150-loop. The NA structural snapshots were

extracted from H0 trajectory every 20 ps, and superimposed onto

the reference open structure. The previously generated 3D-grid

was also superimposed. Those grid points near protein atoms in

the structural snapshots were systematically deleted. Then the

volume of the 150-cavity was calculated by a measurement script

through counting the remaining grid points. The residues which

influence the volume include all the residues from 147 to 152 in

150-loop as well as the nearby residues coming close to the cavity

during the simulation.

Pair wise clustering analysis. Pair wise gromos clustering

analysis (g_cluster) was performed, based on RMSD of 150-loop

[32]. The snapshots to do the clustering analysis were selected at

every 50 ps (total 6,000 snapshots in each system) from H0

trajectory.

Dihedral principal components analysis (dPCA) of 150-

loop. dPCA was performed to describe the conformation

change of 150-loop [33]. Before dPCA, all the backbone dihedral

angles of 150-loop were collected from H0 trajectory from N1o

and N1c system at every 1 ps. With these components prepared,

Table 1. Detailed information of all the MD simulation systems.

system No. of atoms No. of water time (ns) No. of replica

N1o (09N1 with open 150-loop) 35507 9889 300 6

N1c (09N1 with closed 150-loop) 35507 9889 300 6

N2 (N2 with closed 150-loop) 34630 9569 300 6

doi:10.1371/journal.pone.0060995.t001

Figure 1. Representative NA structures together with root mean square deviation of 150-loop in three systems. Root mean squaredeviation (RMSD) of the 150-loop calculated by fitting the whole protein excluding the loop in N1o (A), N1c (B) and N2 (C) systems. Black and red dotsrepresent RMSD value that calculated by fitting to N1 structure with a closed and open conformation of the 150-loop respectively. N1 structure with aclosed and open conformation of 150-loop were shown in (D). Closed and open conformation of 150-loop were colored by cyan and magentarespectively.doi:10.1371/journal.pone.0060995.g001



dPCA was performed, and then the free energy landscape can be

plotted. All the calculated snapshots were projected to the first two

eigenvectors to get a coordinate, in this way to make a two-

dimensional free energy landscape. In this analysis, data of all the

backbone dihedral angles of the 150-loop in both N1o and N1c

systems were dumped together, so that the projections of the

dPCA data from the two systems are comparable.

Transition door way analysis. In order to investigate the

structural transforming pathway of 150-loop, a structural evolution

map was constructed based on all simulation ensembles. In the

transition door way finding, structural snapshot at every 200 ps of

all six trajectories (in total 18,000) were selected to perform

clustering analysis with an RMSD cutoff of 0.25 nm at the first

step. Then all snapshots (time interval 1ps) in six trajectories (in

total 3,600,000) were compared with each cluster centers, if the

RMSD between one snapshot and one cluster center is smaller

than the cutoff (0.25 nm), this snapshot will be assigned to the that

cluster label. Finally, the 150-loop transition door way can be

traced by monitoring the cluster label changes on the time-

continuous replica trajectories.

Results and Discussion

The configuration of 150-loop inter-converted betweenthe open and closed conformations during HREMDsimulations

Figure 1 shows the all atom RMSD of the 150-loop, and it

indicates that this loop inter-converted between the open and the

closed states many times during the HREMD simulations. In N1o

system (Fig. 1A), 150-loop converted to the closed conformation

with small values of RMSD with respect to the closed conforma-

tion (black dots) around 75 ns. Similar structural transforming

behavior was also observed in N1c system (Fig. 1B). The RMSD

values with respect to the open conformation (red dots) decreased

to 0.3 nm around 25 ns, indicating a closed–to-open transition.

Sometimes the RMSD values reached nearly 1 nm. It is because

the RMSD was calculated by superimposing the whole protein

except the 150-loop to reference crystal. Thus, the RMSD values

reflect the overall movement of the loop relative to the other part

of the protein. The large values of RMSD indicate that the

rotational space of the loop relative to the protein is huge. In N2

system, the RMSD values with respect to the putative open

conformation maintained higher than 0.75 nm indicating the

sampled conformations are quite different from the putative open

conformation. On the other hand, RMSD values with respect to

the closed conformation mainly formed two clusters, one around

0.25 nm, and the other around 0.5 nm. This large fluctuation (the

black dots) shows high heterogeneity of the 150-loop of N2.

Volume of 150-cavityThe 150-cavity identified in a crystal structure of group-1 NAs

was suggested to be a binding site for a new kind of antiviral drugs

[12]. The dynamics of 150-loop, however, directly affects the

volume of this cavity. Thus the volume of 150-cavity is also a

sensitive reaction coordinate for the 150-loop conformational

dynamics. The free energy profile of the system, or the potential of

Figure 2. Potential of mean force (PMF) based on volume of150-cavity. PMF calculated based on volume of 150-cavity. Structurerepresenting two local minima together with 16 A3 and 24 A3 is shownas cartoon with green color. As a comparison, crystal structures withclosed and open configuration of 150-loop are aligned together andcolored cyan and green respectively.doi:10.1371/journal.pone.0060995.g002

Figure 3. Open propensity of 150-cavity and backbonedihedral angle distribution of residue 149. Open percentage for150-cavity is calculated based on its volume in three systems (A). Lineswith square, circle and triangle represents N1o, N1c and N2 systemrespectively. Distribution based on psi angle of residue 149 is shown in(B). Dash, dash-dot and solid line represents N1o, N1c and N2 systemrespectively.doi:10.1371/journal.pone.0060995.g003



mean force (PMF) as a function of the volume of 150-cavity is

constructed based on the simulation data with the cavity volume

being calculated by POVME program (see Fig. 2) [31]. The PMF

analysis was based on the ensemble trajectory in which the original

Hamiltonian without any modification was applied.

To have an idea of the size of the 150-cavity, the volumes of

150-cavity of crystal structures of N2 and 09N1 were also

calculated. It is 0 A3 and 8 A3 for N2 and 09N1 with a closed

150-loop, and 112 A3 in open 150-cavity of modeled 09N1

structure. In Figure 2, two local minima with volume around 75

A3 and 180 A3 were identified. These two local minima with large

cavity indicate that open conformation of 150-loop is favorable for

09N1. Representative structures obtained from cluster analyzing

at different locations on the PMF were shown. To identify a cutoff

value for determining whether the 150-cavity is open, each

representative structure is checked manually. The volume size of

16 A3 (also a local maximum on the PMF) is chosen as a threshold

for classifying whether the 150-loop is open.

With the cutoff of 16 A3 at hand, the propensity of open 150-

loop can be calculated. Figure 3A shows the open percentage of

the 150-cavity during HREMD simulation averaged at every

10 ns. Interestingly, both N1o and N1c systems had similarly high

open propensity at the final stage of the simulations with the open

percentage of 64.92% and 64.79% for N1o and N1c system (the

first 50 ns simulation data were discarded). In contrast, the

average open percentage of 150-cavity for the last 250 ns in N2

system is no more than 50%. Moreover, at the end stage of

simulation, open propensity of N2 decreased, fluctuated around

20%. The data indicates that in 09N1 systems, 150-loop prefers to

stay in a conformation with an open 150-cavity, while in the N2

system, 150-loop would rather exhibit a closed conformation.

Backbone psi dihedral angle and side chain orientationof residue 149

The conformational change of the 150-loop is associated with

the motion of backbone dihedral angles of the loop residues. After

checking the backbone dihedral angles of the 150-loop residues,

psi angle (Y) of residue I149 is found to be different between the

closed and open configurations (Table S1). Y of I149 is 240.82uand 125.73u in the closed and open conformation of 150-loop in

09N1 structures, and this value is 229.20u in V149 of N2 crystal

structure. Moreover, the side chain orientation of the residue 149

is also different between the closed and open states of the 150-loop.

In the closed conformation, the side chain of residue 149 points

towards the binding pocket. However, it points away from binding

pocket in the open 150-loop conformation (Fig. 1D). Some work

also stated that the orientation of the residue 149 are different

between group-1 and group-2 NAs [12]. We found that these two

variables, psi angle (Y) and side chain orientation of residue 149,

are highly related (Fig. S1). Thus, Y of residue 149 can be used to

characterize the different shape of the 150-loop. In Figure 3B, the

distribution of the 150-loop based on Y of I149 in all three systems

of the final 100 ns is shown. Both N1 systems evolved to mainly

open states (peak with positive Y value). N2 system, however,

demonstrates the loop favored closed conformation.

Clustering analysis of the conformation of the 150-loopTo get more details of conformations of the 150-loop in the

enhanced sampling simulations, clustering analyses were carried

out. In Figure 4, structures of cluster centers together with the

initial structure of N1o and N1c system were plotted together. In

the crystal structures, the open 150-loop usually turns away from

the binding pocket and the closed loop is close to the binding

pocket (Fig. 1D). For N1o system, the first, third and fourth

clusters are mainly in the open form (Fig. 4A). For N1c system, the

first and second clusters are marked by open loops (Fig. 4B). The

proportion of open 150-loop in N1o and N1c system was 62.6%

Figure 4. Top 4 dominant cluster center structures based on RMSD of 150-loop in three systems. Cluster analysis was based on RMSD ofthe 150-loop. The structures of the top 4 dominant cluster centers are shown with their cluster size in N1o (A), N1c (B) and N2 system (C) respectively.The conformation of the initial open and closed 150-loop is colored magenta and cyan respectively. The structure of each cluster center is shown ingreen color.doi:10.1371/journal.pone.0060995.g004



and 65.3%, and the similar values here indicate the convergence

of the simulations. Conversely, in N2 system the first and third

clusters, contributing 69.4% of the total ensemble, take the closed

conformation (Fig. 4C). The clustering analysis discloses the

detailed conformations of the 150-loop, and the resultant statistics

proves that our HREMD method could provide a convergent

sampling of the loop conformations within the limited simulation

time.

Turn A gauges the conformational changes of the 150-loop

To further characterize the conformational changes of the 150-

loop, dihedral principal components analysis (dPCA) was per-

formed [33]. In this analysis, data of all the backbone dihedral

angles of the 150-loop in both N1o and N1c systems were dumped

together, so that the projections of the dPCA data from the two

systems are comparable. The free energy landscapes of N1o and

N1c systems generated by dPCA are similar to each other (see

Fig. 5). This indicates that the configuration spaces sampled in the

two systems are similar, and the simulations were converged. Four

local minima on the free energy landscapes were identified, and

their representative structures were chosen by clustering analysis

shown on the right panel. The crystal structure of the 150-loop

includes two turns, and we name them as turn A (composed of

residues 147–150) and turn B (composed of residues 150–152, see

Fig. 5). After comparing these representative structures of local

minima with the open and closed state of 150-loop in crystal

structures, the position and shape of turn B are found to undergo

nearly no changes. It is not the case for turn A. The 150-loop

experienced the conformational transitions, from open to closed

conformation, and vice verse, through altering the position and

shape of turn A. Thus, turn A is identified as the structural domain

to gauge the conformational transition of the 150-loop. Taken

together with previous finding, the flexibility of backbone of

residue 149 induces deformation of turn A, and further causes the

150-cavity to open up or to diminish. Similar results were also

observed from N2 dPCA analysis (Fig. S2).

Figure 5. Dihedral PCA indicated turn A of 150-loop is dominant changing place. Free energy landscape obtained from dihedral PCAanalysis of N1o and N1c system is shown in panel (A) and (B) respectively. Four local minima were highlight and their representative structures wereshown on its right panel by clustering analysis. Structure of cluster 1, 2, 3, 4 is shown in forest green, green, lemon and yellow color. Crystal structurewith closed and open 150-loop is shown in cyan and magenta color respectively.doi:10.1371/journal.pone.0060995.g005



Salt bridge of 150-loop in N2 systemIn group-2 NAs, there is an atypical interaction of N2 with a

D147-H150 salt bridge in the 150-loop. This salt bridge was

believed to lock the 150-loop in the closed conformation, and to

control the formation of 150-cavity [16]. With the current

enhanced sampling technique applied, multiple events of breaking

and formation of the salt bridge happened. Interestingly, in the

HREMD simulation, it is found that the probability of salt bridge

formation does not have correlation with the open propensity of

the 150-loop gauged by the volume of the 150-cavity (Fig. 6A).

The structure snapshots shown in Figure 6B and 6C were

obtained from the clustering analysis in the first and last 100 ns

simulations. Structures in Figure 6B show that the 150-loop is in

the open conformation while the salt-bridge still formed (snapshot

colored by orange and light blue) and in Figure 6C the 150-loop is

in the closed conformation while most of the salt-bridge are lost

(snapshot colored by orange and yellow). Our finding here is at

odd with the previous belief [16]. The reason may be that in the

normal MD simulations, the salt bridge rarely broke resulting in a

limited sampling.

Thus the reason why the N2 loop prefers a closed conformation

cannot be easily explained by the presence of the salt-bridge within

150-loop. In Figure 7A, the correlation between contact number

of two loops and open propensity of 150-cavity in both strains

indicates that the intimate interaction between 150 and 430 loops

renders 150-loop in a closed form. The dynamics of 430-loop in all

three systems were investigated by calculating backbone and all

atom RMSD. The backbone of 430-loop in all systems remained

stable. However, the relatively larger values of all atom RMSD of

430-loop of N2 system than those of N1 systems indicated that

430-loop is more flexible in N2 system (Fig. S3). Compare the

minimal distance distribution between two loops in both strains,

N2 had a completely different distribution (Fig. 7B). After

clustering analysis of the ensemble that contributes the highest

distribution peak in N2 system, a salt bridge interaction between

150 and 430-loop was discovered (Fig. 7D). In N2 strain, when the

salt bridge within 150-loop breaks, the side chain of negative

charged D147 turns away and interacts with R430 in 430-loop.

This intimate interaction cannot form in N1 strain (Fig. 7C) due to

the sequence difference between two groups of NAs. To have a

check of the conservation of the salt bridge interaction between

D147 and R430 in group-2 NAs, a multiple sequence alignment

analysis was performed, and the alignment result was shown in

sequence logo format in Figure S4. The co-occurrence of both

D147 and R430 is found with a proportion of 88.04% and 53.58%

in N2 strain of homo and all species respectively. This high

occurrence proportion of D147 and R430 in N2 indicates that the

salt bridge interaction between D147 and R430 has a non-

accidental contribution to stabilize the closed conformation of 150-

loop.

Transition doorwayTo investigate detailed transition pathways of the 150-loop of

NA in 09N1, a structure evolution map was constructed based on

conformation ensemble of 09N1 systems, 3,600,000 structure

snapshots in total. Firstly, structures at every 200 snapshots (in

total 18,000) were selected to perform clustering analysis with an

RMSD cutoff of 0.25 nm. The first largest 200 clusters which

represent more than 80% of the chosen ensemble were picked out.

The cluster centers were considered as the reference structures.

Secondly, based on the RMSD values, all 3,600,000 structures

were assigned to a member of the 200 clusters, if the RMSD of the

150-loop with respect to a certain reference structure smaller than

0.25 nm. Finally, the 150-loop transition was represented by

monitoring the cluster label changes on the time-continuous

replica trajectories. The transitions happened more than 100 times

are shown in Figure 8. Because structures with closed conforma-

tion of the 150-loop only accounted for a small part of the

simulation ensemble of 09N1 systems, this transition pathways did

Figure 6. Correlation between salt bridge forming propensityand open percentage of 150-cavity. Correlation between thepropensity of salt bridge forming and open percentage of the 150-loopbased on volume calculation is shown in (A), both the open percentagebased on volume calculation and salt bridge forming propensity wereaverage at every 1 ns. Structures of cluster centers in the first 100 nsand the final 100 ns of N2 system are shown in panel (B) and (C). Thecrystal structure of N2 is shown in green color, and structure snapshotsfrom simulations are shown in pink, yellow, orange and palecyan color.doi:10.1371/journal.pone.0060995.g006



not catch the transition path between the closed and close-meta

states of the 150-loop. In Figure 8, transition pathway of the 150-

loop is shown as ‘‘close-meta’’ (green color)–‘‘open-meta’’ (red

color)–‘‘open’’ (orange color)–‘‘largely open’’ (yellow color) states

of the 150-loop.

In Figure 8B, an example of the 150-loop conformational

changing event is shown using 6 structures with different states of

the 150-loop, it is noteworthy that the representative structures

here are corresponded to cluster centers that plot Figure 8A. By

altering the position and shape of turn A, conformation of 150-

loop is changing from the closed state to the open state. This

transition feature is consistent with the result obtained from dPCA

analysis, which shows turn A is the dominant changing motif of the

150-loop. In addition, the side chain of I149 also changes

orientation from pointing to the binding pocket (pointing upward

in Fig. 8B) to other directions.

Conclusion

In this study, a Hamiltonian replica exchange molecular

dynamics method was applied to study the dynamic of the 150-

loop in NAs. The crystal structure of 2009 pandemic N1 (09N1)

lacks the 150-cavity. In our HREMD simulations, however, based

on the PMF as a function of the volume of 150-cavity, open

conformation of the 150-loop is found to be more free energy

favorable in this 09N1 structure. Comparing to 09N1, the 150-loop

of N2 prefers to stay in a closed configuration. We do not find

evidences to support the D147–H150 salt bridge as a major factor

for stabilizing the closed conformation. Instead, the more intense

salt bridge interaction between the 150 and 430 loops in N2 system

than in N1 accounts for the biased conformation distribution. The

dPCA analysis together with transition doorway analysis discovered

that the turn A (residues 147–150) of the 150-loop in 09N1 is the

most dynamic motif. By altering its conformation and position,

different conformation of 150-loop could inter-convert among one

another. In further drug discovery studies, the potential drug

molecule can be designed specifically to interact with turn A, in this

way to target at one particular conformation of 150-loop. Besides,

side chain orientation of I149 of NA is also an important character

which highly relates with different conformation of the 150-loop.

In this HREMD sampling scheme, the modified Hamiltonians

only affect the 150-loop and neighboring residues. The number of

replicas needed, thus, can be as low as six. On the other hand, the

other part of the protein will experience few perturbations from

the modified energy functions. The sampling efficiency of that part

is the same as in normal MD simulations. The comparison of

residue-wise root mean square fluctuation (RMSF) between

HREMD and normal MD was performed and the related

discussion was added to the Support Information (Fig. S5). RMSF

values of the 150-loop in HREMD are larger than those of normal

Figure 7. Correlation between open propensity of 150-cavity and contact number between 150 and 430 loops. Correlation betweenopen percentage based on volume and contact number between 150 and 430-loop of two strains is shown in (A), both the open propensity of 150-cavity and contact number between 150 and 430-loop were averaged at every 10 ns. Distribution of minimal distance between heavy atoms of 150and 430-loop is shown in (B). Panel (C) and (D) represent structure of the highest peak in N1 and N2 in (D) respectively.doi:10.1371/journal.pone.0060995.g007



MD simulation, and this phenomenon is as expected. The overall

RMSF patterns are quite similar between the two data sets

indicating that our HREMD scheme can selectively enhance

sampling of 150-loop dynamics. If the dynamics of the 150-loop is

strongly coupled with parts of protein which are located far, such

as in the case of allosteric regulation [34], the sampling efficiency

will be limited. Based on the data obtained, the motion of 150-loop

is quite decoupled from other parts of the protein, which is an ideal

paradigm for application of such HREMD method. From the

methodological point of view, such HREMD method is similar to

the locally enhanced sampling method [35], however, the latter

usually cannot guarantee the output as a canonical ensemble.

The dynamics of the 150-loop has been found to be critical in

mediating drug-protein interaction [36], [37] and drug resistance

[37], [38]. Opening of the 150-cavity has become a new target for

new inhibitor design [12], [14]. It is not clear what is the biological

function of the plasticity of the 150-loop. As suggested by Amaro

and coworkers, the opening and closing of the 150-cavity may be

required for natural sialoglycan substrates to fit into the active site,

given the bulky nature of these glycans [16]. Landon et al. used

MD simulations and computational solvent mapping (CS-Map)

method to identify that 150 and 430-loops are the novel druggable

hot spots region [39]. Cheng et al. performed docking study on the

crystal N1 structure as well as cluster representative structures

from MD simulation, and found that half of their top hits would be

neglected if only based on the crystal structure. Interestingly, these

top hits have the preference to bind with the flexible 150 and 430

loops [40]. All the studies indicated the importance of identifying

different conformations of NA, especially of the 150 and 430-loop

region. Moreover, Cheng et al. have proposed the interaction

pattern of the compound that docked into both 150 and 430-

cavity. Grienke et al. have already identified a T-shaped

compound that has inhibition effect and may interact with NA

on an open 150-cavity [40], [41]. Our current study has made

efforts in exploring in more depth the dynamic properties of the

loop that provides more information for further drug discovery.

This molecular insight will open new scenery to derive more

potent ligands with novel scaffolds specifically targeting on the

flexible 150-loop. Our group is currently pursuing in this direction.

Supporting Information

Figure S1 Correlation between open percentages calcu-lated by side chain orientation of residue 149 anddihedral angle psi (Y) of residue 149 in three systems.Open propensity based on psi of 149 is calculated as that, if this

angle is smaller than 45u and larger than 2135u, the 150-loop can

be considered as closed, otherwise, it is open. Similarly, if side

chain of residue 149 points towards binding pocket, it will be

considered as closed, otherwise, 150-loop is open. Black, red and

blue color line represents N1o, N1c and N2 system respectively.

(TIF)

Figure S2 Dihedral PCA analysis of 150-loop in N2system. Free energy landscape of shown in panel A, three local

minima were highlight and their representative structures were

shown on its right panel (B) by clustering analysis. Structure of

cluster 1, 2, 3 is shown in green, pale green and forest color.

Structure with closed and open 150-loop is shown in cyan and

magenta color respectively.

(TIF)

Figure S3 Backbone and all atom RMSD of 430-loop inall three systems. The backbone and all atom RMSD of 430-

loop is shown in black and red dot respectively.

(TIF)

Figure S4 Multiple sequences alignment and sequencelogo of 150 and 430 loops for N2 strain. Panel A and B

shows the sequence alignment within homo and all species of N2

strain for influenza virus.

(TIF)

Figure S5 The residue-wise root mean square fluctua-tion (RMSF) compared between HREMD N1c systemand normal MD. RMSF compared between HREMD N1c

system (red curve) and normal MD (black curve). Data of normal

MD came from one of our previous work [38].

(TIF)

Table S1 Backbone dihedral angle of residues in 150-loop in 09N1 systems.

(DOC)

Figure 8. Conformational transition doorway of 150-loop in09N1. Conformational transition doorway of 150-loop in 09N1 (A), onlythose with transition times larger than 100 are displayed. Symbols inyellow, orange, red and green color represent largely open, open, open-meta and close-meta conformation of 150-loop respectively; Structuresnapshots with different conformation of 150-loop relating the symbolsin the transition doorway (B). The initially open and closed 150-loop iscolored magenta and cyan respectively. Each cluster center structure isshown in green color. ILE149 is shown as sticks.doi:10.1371/journal.pone.0060995.g008



Acknowledgments

This work is partially supported by the IDA Cloud Computing Call for

Project Proposals 2012 and MOE NTU Tier 1 grant RG23/11. We also

thank Ping Jiang for her helpful discussions.

Author Contributions

Conceived and designed the experiments: YM. Performed the experiments:

NH YM. Analyzed the data: NH YM. Contributed reagents/materials/

analysis tools: NH YM. Wrote the paper: NH YM.

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